Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Apical dendrite wikipedia , lookup
Subventricular zone wikipedia , lookup
Premovement neuronal activity wikipedia , lookup
Clinical neurochemistry wikipedia , lookup
Neuroregeneration wikipedia , lookup
Optogenetics wikipedia , lookup
Development of the nervous system wikipedia , lookup
Circumventricular organs wikipedia , lookup
Neuroanatomy wikipedia , lookup
Microneurography wikipedia , lookup
Basal ganglia wikipedia , lookup
Tr. J. of Veterinary and Animal Sicences 23 (1999) 153-158 © TÜBİTAK The Location of Extrinsic Afferent and Efferent Neurons Innervating The Stomach and Colon in Rat Berrin TARAKÇİ University of Fırat, Faculty of Veteriner Medicine, Elazığ-TURKEY Camille VAİLLANT University of Liverpool, Veterinary Science, Liverpool-ENGLAND Received: 23.01.1998 Abstract: The location of extrinsic afferent and efferent neurons projecting to the rat gastrointestinal tract were mapped using the retrograde tracer, True Blue (TB). Injection of retrograde tracer, TB into stomach, proximal colon and distal colon resulted in labelled cells in the dorsal root ganglia. Cells were also labelled in the nodose ganglia following injection of tracer into stomach and proximal colon. TB-labelled cells were also seen in the prevertebral ganglia (coeliacomesenteric complex and inferior mesenteric ganglion), after injection of TB into the stomach, proximal and distal colon. Many TB-labelled cells were detected in the dorsal motor nucleus of the vagus (DMNX) following injection of tracer into stomach. Labelled cells were less numerous following injection of tracer into the distal colon. Key Words: Retrograde tracer, afferent neuron, efferent neuron, stomach, colon , rat. Ratlarda Mide ve Kolonu İnnerve Eden Ekstrinsik Afferent ve Efferent Nöronların Lokalizasyonu Özet: Ratlarda mide ve kolona uzantı gönderen ekstrinsik afferent ve efferent nöronların lokalizasyonu retrograde tracer, True Blue (TB) kullanılarak araştırıldı. Tracer’in mide ile kolonun başlangıç ve son bölümlerine enjeksiyonu sonrası spinal ganglionda TB ile işaretlenmiş hücrelere rastlandı. Tracer’in mide ve kolonun başlangıcına enjeksiyonu sonrası vagal ganglionda işaretlenmiş hücreler gözlendi. TB’nin mide, kolonun başlangıç ve son bölümlerine enjeksiyonunu takiben prevertebral ganglionda (coeliacomesenteric complex ve inferior mesenteric ganglion), TB ile işaretlenmiş hücrelere rastlandı. Tracer’in mide’ye enjeksiyonu sonrası çok sayıda, kolonun başlangıç kısmına enjeksiyonu sonrası ise vagusun dorsal motor nükleusunda (DMNX) az sayıda işaretlenmiş hücreler gözlendi. Tracer’in distal kolona enjeksiyonu sonucu ise DMNX ve sakral medulla spinalis’de TB ile işaretlenmiş hücrelere rastlanmadı. Anahtar Sözcükler: Retrograde tracer, afferent nöron, efferent nöron, mide, kolon, rat. Introduction The innervation of the gastrointestinal tract consists of a vast number of nerve cell bodies and their processes embedded in the wall of the gut, together with an input from extrinsic sources (1-4). Early physiological studies clearly demonstrated the presence of extrinsic fibres projecting to the gut (5-12). However, precise identification of the cells involved awaited the development of retrograde tracing techniques, involving the pinocytotic uptake of a substance at nerve terminals, its transport back to the cell body, and its subsequent visualisation (13-21). In the present study, the origins of afferent and efferent fibres projecting to stomach, proximal and distal colon were examined using fluorescence retrograde tracing technique. Materials and Methods Retrograde tracing techniques Adult twelve wistar rats were anaesthetised with hypnorm (0.3 ml/kg, i.m.) and diazepam (2.5 mg/kg, i.p.). Using aseptic techniques suspension of True Blue in distilled water (5% w/v) was injected into the ventral wall of stomach, into the proximal colon or into the distal colon with 10 µl Hamilton microsyringe. A total of 20 µl, in volumes of 1-2 µl, was injected into each region. After each injection, the needle was left in place for up to 1 minute to reduce leakage of dye long the needle tract, and injection site was then swapped with saline. Viscera were replaced in the abdominal cavity, and the incision in the abdominal muscle, then skin was sutured. Tissue processing Seven days after injection, rats were terminally 153 The Location of Extrinsic Afferent and Afferent Neurons Innervating The Stomach and Colon in Rat Table 1. Table 2. Percentage of True Blue-labelled spinal afferents (mean±S.D.), after injecting TB into the stomach (left and right DRGs, segments T9-L3, 3 rats). Ganglia % T 24.1±4.7 9 T10 19.8±1.6 T11 24.7±2.5 T12 17.1±2.1 T13 9.9±6.7 L1 4.1±2.4 L2 1.2±0.1 L3 0.8±0.1 Percentage of True Blue-labelled spinal afferents (mean±S.D.), afternjecting TB into the proximal colon (left and right DRGs, segments T10-S3, 6 rats). Ganglia T 10 % 19.5±3.8 T11 26.3±6.5 T12 22.4±1.9 T13 21.4±3.6 L1 8.1±3.3 L2 2.5±1.9 L3 0.8±0.1 L4 0 L5 0 L6 0 S1 0 S2 0 S3 0 anaesthetised (overdose of sodium pentobarbitone, i.p.) and 0.1 ml of heparin (5000 I.U. per ml) was injected into the left ventricle of the heart. The animals were transcardically perfused with PBS (0.1M phosphate 154 buffer, 0.14M sodium chloride, pH 7.4) followed by 4% formaldehyde in sodium cacodylate buffer (0.1M, pH 7.4). Nodose ganglia, dorsal root ganglia, brain stem, spinal cord (segment T9-10 and S1-2), celiac, splanchnic and inferior mesenteric ganglia were removed. Tissues were placed in 0.1M sodium cacodylate, pH 7.4, containing 20% sucrose for at least 24 hours at 4˚C before snap freezing and sectioning at 10-15µm in a cryostat. Sections were examined using a Leitz Dialux 20 microscope. Labelled cells were observed using Leitz excitation-emission filter pack A for True Blue. Results Extrinsic afferents Injection of retrograde tracer, TB into stomach, proximal colon and distal colon resulted in labelled cells in the dorsal root ganglia. Cells were also labelled in the nodose ganglia following injection of tracer into stomach and proximal colon (Figs. 1 & 2). Gastric afferents In dorsal root ganglia, TB labelling was bilateral and, in any one segment, left and right ganglia contained similar number labelled cells. Higher numbers were seen in ganglia of segments T9-T12, the number of labelled cells declined rapidly in lumbar ganglia (Table 1). Labelling in the nodose ganglia was also bilateral, but the left ganglia contained more TB-labelled cells than the right. TB-labelled cells were concentrated around the caudal pole of the ganglion. Intestinal afferents Spinal projections to the proximal colon extended mainly from T10 through to L2. Highest numbers of TBlabelled cells were seen in ganglia of segments T10-T13, numbers of labelled cells declined rapidly in lumbar segments (Table 2). Vagal afferents projecting to the proximal colon were detected bilaterally, they were much fewer in number compared to vagal gastric afferents. Spinal projections to the distal colon werefound mainly in the thoracolumbar segments T11-L2, and in the first sacral segment, with the majority of labelled cells lying in segments L1-L2 and S1 (Table 3). No vagal projection to the distal colon could be detected. Extrinsinc efferents Injections of TB into the stomach, proximal and distal B. TARAKÇI, C. VAILANT Table 3. Percentage of True Blue-labelled spinal afferents (mean ±S.D.), after injecting TB into the distal colon (left and right DRGs, segments T10-S3, 6 rats). Ganglia T Figure 1. 11 % 10.9±5.9 T12 9.2±2.3 T13 9.1±1.3 L1 25.8±2 L2 21.1±9.7 L3 1.3±0.8 L4 0 L5 0 L6 0 S1 21.5±9.5 S2 0 S3 0 TB labelled cells in dorsal root ganglion from a rat injected with TB in the ventral wall of the stomach. 230x. colon resulted in TB-labelled cells in the prevertebral ganglia, that is, the coeliacomesenteric complex and inferior mesenteric ganglion (Fig. 3). Following injection of TB into stomach and proximal Figure 2. TB labelled cells in nodose ganglion from an rat injected with TB in the wall of the proximal colon. 230x. Figure 3. TB labelled cells in inferior mesenteric ganglion from a rat injected with TB in the wall of the distal colon. 300x. colon, there was a distinctive distribution of labelled cells in the coeliacomesenteric complex. Within the fused coeliacomesenteric ganglia, moderate numbers of labelled cells were scattered throughout the tissue. However, as the splanchnic ganglia, moderate numbers of labelled cells were scattered throughout the tissue. However, as the splanchnic nerves approached the coeliacomesenteric ganglion, there were numerous cell bodies in the nerve trunks of which a large proportion were labelled. In the text these regions are termed the splanchnic ganglia, whereas the remaining part of the complex, the coeliacomesenteric ganglion, is abbreviated to coeliac ganglion. Following injection of tracer into the stomach, labelled cells were found mainly in the splanchnic and coeliac 155 The Location of Extrinsic Afferent and Afferent Neurons Innervating The Stomach and Colon in Rat This distribution of spinal afferents projecting to the distal colon is in agreement with data from other studies on the rat (18, 19). Baron and Janig (24) showed that application of tracer to lumbar colonic nerves labelled cells in dorsal root ganglia mainly in lumbar segments L1 and L2. Thus, it appears that most of the spinal afferents projecting to the distal colon do so via the inferior mesenteric ganglion and lumbar colonic nerves, whereas those projecting to the proximal colon clearly take a different route. Figure 4. TB labelled cells in dorsal motor nucleus of the vagus (DMNX) of a rat injected with TB in the wall of the proximal colon. 300x. ganglia. Very few cells were detected in the inferior mesenteric ganglion. Injection of tracer into the distal colon resulted in labelling of cells mainly in the coeliac and inferior mesenteric ganglion, with few cells labelled in the splanchnic ganglia, whereas after injection into the proximal colon labelled cells were found in all three tissues. Many TB-labelled cells were detected in the dorsal motor nucleus of the vagus (DMNX) following injection of tracer into stomach. Labelled cells were less numerous following injection of tracer into the proximal colon (Fig. 4). Almost no labelled cells were seen in the DMNX or sacral spinal cord following injection of tracer into the distal colon. Discussion Afferent neurons The distribution of gastric afferents in dorsal root ganglia observed in the present study is in agreement with earlier reports of spinal afferents projecting to the stomach of the rat, guinea pig and cat (14-18). Similarly, the restriction to the caudal pole of the nodose ganglion of vagal afferents projecting to the stomach has been reported previously (22, 23). In the present study, a different pattern was seen in the sensory projections to proximal and distal colon. Spinal projections to the proximal colon were found mainly in thoracic segments T10-T13, whereas those projecting to the distal colon were found mainly in lumbar segments L1 and L2 and in the first sacral segment. 156 A substantial number of cells in the coeliac complex project to the proximal colon (this study, 25) via the inferior coeliac nerves (25), and it is probable that primary afferents projecting to the proximal colon from thoracic ganglia follow this more cranial route. Although, Altschuler et al. (20) detected labelled afferent nerve terminals in the nucleus of the solitary tract following injection of retrograde tracer into the rat colon, no study until now has demonstrated nodose ganglion cells projecting to the colon. In the present sutdy, labelled cells were detected bilaterally following injection into the proximal colon, but they wereuch fever in number than vagal afferents projecting to the stomach. In the study by Altschuler et al. (20), labelled fibres were detected in the nucleus of solitary tract in one out of five animals that had received injection of cholera toxinperoxidase tracer into the distal (descending) colon. In contrast, in the present study, no vagal afferents projecting to the distal colon could be detected, which is probably due to difference in the retrograde tracer employed or to reflection of the more distally restricted site of injection used here. Efferent neurons As observed in the present study, retrograde labelling studies in various species have demonstrated that the stomach in innervated by parasympathetic pregnanglionc neurons from the dorsal motor nucleus of the vagus (DMNX) (14, 15, 18, 26-28). The DMNX also projects to the proximal colon as has been previously shown in rat (20), cat (13) and dog (29), with a marked decline in the number of cells projecting to distal colon. Although labeled cells have been detected in the sacral spinal cord after injection of tracer into the distal region of colon in mouse (30), cat (13), rabbit (31) and dog (29), extremely few cells were detected in the present study on the rat, suggesting only a limited parasympathetic innervation of this region. In support of this, Altschuler et al. (20) found very few labelled cells in the sacral spinal cord in only one out of five rats receiving injection of cholera toxin-peroxidase tracer into the distal B. TARAKÇI, C. VAILANT colon and relatively few labelled cells in the DMNX. An alternative explanation for absence of label in sacral preganglionic parasympathetic neurons is that these noeurons project to the distal colon largely via second order neurons in pelvic ganglia, as is seen in the dog (29), rather than projecting directly to the gut. It is not known if these ganglia exist in the rat. In the dog they could not be identified at dissection, and were only recognised at light microscopic level by the presence of labelled cells. Further retrograde labelling studies are required to explore this posibility. It is well established that the coeliac ganglion sends projections mainly to the stomach and small intestine, with a minor projection to the proximal colon, whereas the large intestine receives its sympathetic innervation mainly from the inferior mesenteric ganglion (18, 23, 25). The results of the present study are in general agreement with there fidings, but include evidence of projections from the splanchnic ganglia to stomach and colon. In an early report, Green (32) described the coeliac complex as a single ganglion, but later studies revealed multiple components, namely suprarenal, aorticorenal and coeliac ganglia, but no separate superior mesenteric ganglia (33, 34, 35). The splanchnic ganglia described in the present study are identical in position to the suprarenal ganglia, so named by Baljet and Drukker (33) primarily on the grounds of their anatomical position, although they did stress projections from the ganglia to the adrenal glands. Similar ganglia in man have been described as splanchnic ganglia of Lobstein (36). While these ganglia within the splanchnic nerves may project to the adrenal glands, the present study has demonstrated that they have a much wider territory, that is, the gastrointestinal tract. Thus, it seems more appropriate to apply the term ‘splanchnic’, rater than ‘suprarenal’, to these ganglia. References 1. Gabella G Innervation of the gastrointestinal tract. In Rev Cytol (1979) 59: 129-193. 2. Gershon MD and Erde SM The nervous system of the gut. Gastroenterology (1981) 80: 1571-1594. 3. 4. 5. Szurszewski JH and King BF Physiology of prevertebral ganglia in mammals with special reference to inferior mesenteric ganglion. In: Handbook of Physiology, Sections 6: The Gastrointestinal System. Vol. 1, part 1, eds. SG Schultz, JD wood and BB Rauner. American Physiological Society, Bethesda. pp. 519-592 (1989). Furness JB and Bornstein JC The extrinsic nervous system and its extrinsic connections. In the: Textbook of Gastroenterology. Volume 1, eds: T Yamada, DH Alphers, C Ownyang DW Powell and FE Silverstein. Lippincott Company, Philadelphia. pp. 2-24 (1991). Bayliss WM and Starling AE The movements and innervation of the small intestine. J Physiol (1899) 24: 99-143. 6. Bayliss WM and Starling AE The movements and innervation of the small intestine. J Physiol (1900a) 26: 127-138. 7. Bayliss WM and Starling AE The movements and innervation of the large intestine. J. Physiol (1900b) 26: 107-118. 8. Elliott TR and Barclay-Smith E Antiperistalsis and other muscular activities of the colon. J Physiol (1904) 31: 272-304. 9. McSwiney BA Innervation of the stomach. Physiol Rev (1931) 11: 478-514. 10. Kewenter J The vagal control of the jejunal and ileal motility and blood flow. Acta Physiol Scand. Suppl (1965) Vol 65. Suppl 251, pp.5-65. 11. Hulten L Extrinsic nervous control of colonic motility and blood flow. Acta Physiol Scand. Suppl (1969) 335, pp. 9-108. 12. Rostad H Colonic motility in the cat. Acta Physiol Scand (1973) 89: 91-103. 13. Satomi HT, Yamamoto H and Takatama H Origins of parasympathetic preganglionic fibres to the cat intestine as demonstrated by the horseradish peroxidase method. Brain Res (1978) 151: 571-578. 14. Kalia M and Mesulam MM Brain stem projections of senbory and motor components of the vagus complex in the cat. II. Laryngeal, tracheobronchial, pulmonary, cardiac and gastrointestinal branches. J Comp Neurol (1980) 193: 467-508. 15. Shapiro RE and Miselis RR The central organisation of the vagus nerve innervating the stomach of the rat. J Comp Neurol (1985) 238: 473-488. 16. Green T and Dockray GJ Calcitonin gene-related peptide and substance P in afferents to the upper gastrointestinal tract in the rat. Neurosci Lett (1987) 76: 151-156. 17. Green T and Dockray GJ Characterisation of the peptidergic afferent innervation of the stomach in the rat, mouse and guinea pig Neuroscience (1988) 25: 181-193. 157 The Location of Extrinsic Afferent and Afferent Neurons Innervating The Stomach and Colon in Rat 18. Shu S, Ju G and Fan L The glucose oxidase -DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci Lett (1988) 85: 169-171. 27. Pagani FD, Norman WP, Kasbekar Dk and Gillis RA Localisation of sites within dorsal motor nucleus of vagus that affect gastric motility. Am J Physiol (1984) 249: G73-G84. 19. Keast JR and De-Groat WC Segmental disribution and peptide content of primary afferent neurons innervating the urogenital organs and colon of male rats. J Comp Neurol (1992) 319: 615623. 28. Norman WP, Pagani FD, Ormsbee HS, Kasbekar DK and Gillis RA Use of horseradish peroxidase to identify ihndbrain sites which influence gastric motilitiy in the cat. Gastroenterology (1985) 88: 701-705. 20. Altschuler SM, Escardo J, Lynn RB and Miselis RR The central organisation of the vagus nerve innervating the colon of the rat, Gastroenterology (1993) 104: 502-509. 29. Hudson LC Horseradish peroxidase study of the location of extrincis efferent and afferent neurons innervating the colon of the dog. Am J Vet Res (1990) 51: 1875-1881. 21. Ali AHM Studies on the sensory innervation of the rat stomach. (1994) PhD Thesis. 30. 22. Sharkey KA, Williams RG and Dockary GJ Sensory substance P innervation of the stomach and pancreas: demonstration of capsaicin-sensitive sensory neurons in the rat by combined immunohistochemistry and retrograde tracing. Gastroenterology (1984) 87: 914-921. Payette R, Tennyson V, Pham T, Mawe G, Pomeranz H, Rothman T and Gershon MD Origin and morphology of nerve fibres in the aganglionic colon of the lethal spotted mutant mouse. J Comp Neurol (1987) 257: 237-252. 31. Bessant ARD and Robertson-Rintoul J Origin of the parasympathetic preganglionic fibres to the distal colon of the rabbit as demonstrated by the horseradish peroxidase method. neurosci Lett (1986) 63: 17-22. 32. Green EL The anatomy of the rat. Hafner, New York. (1963) 33. Baljet B and Drukker J The extrinsic innervation of the abdominal organs in the female rat. Acta anat (1979) 104: 243-267. 34. Hamer D and Santer R Anatomy and blood supply to the coeliacsuperior mesenteric ganglion complex of the rat. Anat Embryol (1981) 162: 353-362. 35. Berthoud HR and Powley TL Characterisation of vagal innervation to the rat celiac, suprarenal and mesenteric ganglia. J Auton Nerv Syst (1983) 42: 153-169. 36. Lobstein JF De nervi sympathetic humani fabrica. Levrault, Paris. (from Baljet and Drukker, 1979, Acta Anatomica (1823) 104: 243-167. 23. Green T Studies on the organisation and function of the afferent innervation of the upper gastrointestinal tract. (1988) PhD Thesis. 24. Barron R and Janig W AFferent and sympathetic neurons projecting into lumbar visceral nerves of the mael rat. J Comp Neurol (1991) 314: 429-436. 25. Kreulen DL and Szurszewski Electrophysiological and morphological basis for organisation of neurons in prevertebral ganglia. In the: Frontiers of Knowledge in the Diarheal Diseases: International Colloquium in Gastroenterology, eds. HD Janowitz and DB Sachor. Upper Montclair, NJ: Projects in Health, pp.211226 (1979). 26. 158 Yamamoto T, Carr PA, Baimbridge KG and Nagy JI Parvalbuminand calbindin D28k- immunoreactive neurons in the superficial layers of the spinal cord dorsal horn of rat. Brain Res Bull (1989) 23: 493-598.